Animals across diverse phyla can regenerate lost structures, a capacity that is considerably more limited in mammals. Several chordate species including urodele salamanders and teleost fish can regenerate appendages and solid organs, yet among mammals such adult organogenesis is rarely—if ever—observed. An important exception is wound-induced hair neogenesis (WIHN), a phenomenon in which skin and hair follicles are regenerated following large, full thickness wounds in mice or rabbits (Breedis, 1954; Ito et al., 2007) The complete regeneration observed in WIHN is in marked contrast to the fibrotic scarring that typically results from cutaneous wound healing. Regenerated hair follicles are complex mini-organs with disparate cell types, dedicated neurovascular support, and a distinct stem cell compartment located in the bulge region. These stem cells not only repopulate hair follicles throughout life, but also aid in skin re-epithelialization after wounding, pointing to the potential therapeutic relevance of WIHN (Ito et al., 2007). As WIHN represents a rare example of adult organogenesis in mammals, understanding its mechanisms could aid in efforts to regenerate other structures.
While originally described in the 1940s, WIHN has only recently been characterized in morphogenic and molecular detail (Breedis, 1954; Gay et al., 2013; Ito et al., 2007; Kligman and Strauss, 1956; Myung et al., 2013; Nelson et al., 2013). Following complete excision of skin down to fascia, wounds on the backs of mice are closed through initial contracture and then reepithelialization. Subsequently, hair follicle morphogenesis ensues with recapitulation of events that occur during embryonic hair development. Formation and invagination of epithelial placodes in the epidermis, induction of adjacent dermal papillae, and ultimately, elaboration of distinct hair cell subtypes are observed (Ito et al., 2007). Follicle-associated structures such as sebaceous glands are also regenerated. Regenerated follicles transit through multiple hair cycles, just like neighboring hairs from unwounded skin (Ito et al., 2007). Therefore, WIHN represents functional regeneration rather than mere wound repair through scarring.
Developmental pathways required for embryonic organogenesis are reactivated following trauma. In axolotl limb regeneration for example, Shh signaling is activated at the site of injury in the residual limb much as it is induced in the zone of polarizing activity during limb development (Torok et al., 1999). Similarly, during WIHN, signaling pathways utilized in embryonic hair formation reemerge after wounding. Activation of the canonical Wnt pathway is one of the earliest events observed in follicular morphogenesis. Wnt activation occurs around E15 in mice as the undifferentiated epithelium begins to condense into epithelial placodes at sites of future follicle formation (Millar, 2002). Similarly, after cutaneous wounding, the Wnt ligand, Wnt10b, and the Wnt effector, Lef1, are induced after re-epithelialization is complete, but prior to the emergence of new follicles (Ito et al., 2007). Wnt pathway activation is critical for hair morphogenesis during both development and regeneration, as mice deficient in Wnt signaling fail to generate hairs (Ito et al., 2007; Myung et al., 2013). Secondary to Wnt activation during follicular development, Shh signaling is induced in epithelial placodes and underlying dermal papillae. Activation of the Shh pathway contributes to subsequent hair follicle invagination and morphogenesis (St-Jacques et al., 1998). The Shh pathway is similarly induced during adult hair follicle regeneration. Other molecular details of hair regeneration are shared with hair development including expression of the hair cytokeratin Krt17 and activation of alkaline phosphatase activity in dermal papillae (Ito et al., 2007).
While downstream morphogenic events in WIHN parallel those in hair development, the signals triggering reactivation of these programs in adult regeneration are unclear. To initiate regeneration organisms must first sense a loss of tissue integrity. Candidate signals include molecules liberated from damaged tissues as well as mediators released by infiltrating immune cells. In newts and axolotls, activation of thrombin is a key early event in regeneration. Inhibition of thrombin activation abrogates lens regeneration in newts, for example (Imokawa and Brockes, 2003). Recently it has been shown that FGF9 released from γδ T cells several days after wounding promotes hair regeneration in rodents (Gay et al., 2013). However, the most proximal signals released by damaged keratinocytes to initiate regeneration in the skin remain unknown. Discovery of such damage-associated signals may explain why wound healing during WIHN proceeds with regeneration whereas most cutaneous wound healing in mammals leads to fibrotic scarring. Identifying these molecules may also suggest therapeutic approaches to promote skin and hair regeneration and reduce fibrosis.
To identify molecular events that initiate regeneration, we exploited the natural variation in regenerative capacity observed in various mouse strains. Through gene expression screening of healed wounds prior to regeneration, we identified the pattern recognition receptor, Toll-like Receptor 3 (TLR3), as a critical regulator of cutaneous regeneration, as it is up-regulated in highly regenerative mice. We identified dsRNA released from damaged cells as key triggers of the regeneration process through their activation of TLR3. The ensuing damage-induced signaling cascade prevents normal keratinocyte differentiation and promotes the acquisition of stem cell features in keratinocytes. Furthermore, TLR3 activation initiates molecular events in the hair morphogenic program, with activation of canonical Wnt, Shh pathways and EDAR resulting in augmented hair follicle neogenesis. Thus, TLR3 activation by dsRNA links damage sensing after wounding to the earliest molecular events in hair regeneration. These results uncover a novel role for TLR3 as a master regulator of regeneration in the skin.